Phosphorylation of both EGFR and ErbB2 is a reliable predictor of prostate cancer cell proliferation in response to EGF

Abstract

Despite multiple reports of overexpression in prostate cancer (PC), the reliance of PC cells on activated epidermal growth factor receptor (EGFR) and its downstream signaling to phosphoinositide 3'-kinase/Akt (PI3K/Akt/PTEN) and/or mitogen-activated protein kinase (MAPK/ERK) pathways has not been fully elucidated. In this study, we compared the role of EGF-mediated signaling in nonmalignant (BPH-1, PNT1A, and PNT1B) and PC cell lines (DU145, PC3, LNCaP, and CWR22Rv1). EGF-induced proliferation was observed in all EGFR-expressing PC cells except PC3, indicating that EGFR expression does not unequivocally trigger proliferation following EGF stimulation. ErbB2 recruitment potentiated EGF-induced signals and was associated with the most pronounced effects of EGF despite low EGFR expression. In this way, the sum of EGFR and ErbB2 receptor phosphorylation proved to be a more sensitive indicator of EGF-induced proliferation than quantification of the expression of either receptor alone. Both Akt and ERK were rapidly phosphorylated in response to EGF, with ERK phosphorylation being the weakest in PC3 cells. Extrapolation of these findings to clinical PC suggests that assessment of phosphorylated EGFR + ErbB2 together could serve as a marker for sensitivity to anti-EGFR-targeted therapies.

Figure 1

Growth curve of PC cells following EGF stimulation. Serum-starved cultures incubated in the absence (-●-) or presence of ascending concentrations of EGF (-◯-, 0.01 ng/ml; -▲-, 0.1 ng/ml; -x-, 1 ng/ml; -□-, 10 ng/ml) were monitored daily by fluorometric measurement of the rate of reduction of alamarBlue for 5 days following treatment. Error bars represent standard error of the mean.

Figure 2

EGF-induced cell proliferation in prostate cell lines. Cells were serum-starved for 24 hours then treated with fresh serum-free medium or 10 ng/ml EGF. Cells were trypsinized and counted after 4 days of treatment. Changes in cell number in response to EGF were represented as fold increase over control. A two-fold change in cell number equals 2.0 fractional change, whereas 1 represents identity with control cells. Data obtained from three experiments are represented as mean with error bars representing standard error of the mean. *P < .05 was considered significant.

Figure 3

Expression of ErbB receptors in prostate cell lines. Protein lysates from an equal number of cells were fractionated by SDS polyacrylamide gel electrophoresis (PAGE) and Western-blotted with indicated antibodies. Proteins were visualized with HRP-labeled secondary antibody and ECL detection. Equal loading was confirmed by blotting for β-actin.

Figure 4

EGF induces ErbB receptor phosphorylation. CWR22Rv1 (panel A) cells and (panel B) LNCaP cells were incubated in serum- free medium overnight then stimulated with 100 ng/ml EGF for 10 minutes at 37C. Cell lysates were prepared with protein A Sepharose beads in the absence (-) or presence of ErbB antibodies or PY20. The resultant immune complexes were fractionated by SDS-PAGE and Western-blotted. The pattern of ErbB phosphorylation shown in CWR22Rv1 (panel A) was typical of that seen in other EGFR-expressing PC cell lines (except LNCaP cells).

Figure 5

EGF induces strong tyrosine phosphorylation in responsive prostatic cells. Panel A: Confluent cultures of prostatic cells were serum-starved overnight then incubated in the absence (-) or presence (+) of EGF (100 ng/ml) for 10 minutes before lysates were prepared. These were immunoprecipitated with antiphosphotyrosine antibody (PY20) and the resultant immune complexes were fractionated by SDS-PAGE and Western-blotted using the antiphosphotyrosine antibody (PY99), EGFR (panel B), and ErbB2 (panel C). Radiographs shown in panels B and C were scanned and digitalized to semiquantify EGFR and ErbB2 phosphorylation (panel D).

Figure 6

Expression of PI3K/Akt/PTEN in prostate cell lines. Protein lysates from an equal number of cells were fractionated by SDS-PAGE and Westernblotted with indicated antibodies. Proteins were visualized with HRP-labeled secondary antibody and ECL detection. Equal loading was confirmed by blotting for β-actin.

Figure 7

Inhibition of PI3K/Akt signals in prostate cell lines. Panels A and B: Indicated cells were serum-starved overnight then incubated with medium (-) or wortmannin (W) (50 nM) for 20 minutes followed by EGF (E) or EGF + more wortmannin (EW). Lysates from stimulated cells were included as a positive control (P). Panel C LNCaP cells were treated with indicated doses of LY294002 followed by EGF stimulation (10 ng/ml). Wortmannin and LY294002 reduced Akt phosphorylation but had no effect on total Akt expression levels. Panel D: PC cells were serum-starved overnight then incubated in the absence (-) or presence of 10 ng/ml EGF (E) for 10 minutes at 37C. Lysates were probed for phosphorylated Akt, phosphorylated MAPK/ERK, and β-actin as a loading control.